Glucagon-like peptide 1 receptor agonists and uses thereof

ABSTRACT

The present invention provides modified glucagon-like peptide 1 (GLP1) polypeptides, fusion proteins comprising modified GLP1 polypeptides, and methods of use thereof. In various embodiments of the invention, the fusion proteins are GLP1 receptor agonists that comprise a modified GLP1 fused to a stabilizing domain. In some embodiments, the fusion proteins comprising modified GLP1 are useful for treating or ameliorating a symptom or indication of a disorder such as obesity and diabetes.

This application is a divisional of U.S. patent application Ser. No.16/137,662 filed Sep. 21, 2018 (now U.S. Pat. No. 11,045,522), whichclaims the benefit of priority to U.S. Provisional Patent ApplicationNo. 62/562,283 filed Sep. 22, 2017, the disclosures of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to human glucagon-like peptide 1receptor agonists, and therapeutic methods of using said agonists.

BACKGROUND OF THE INVENTION

Obesity has become a major health issue in the United States with twoout of three Americans considered to be overweight or obese. Obesity isan important underlying risk factor for developing other diseases suchas heart disease, stroke, and diabetes. Even a modest decrease in bodyweight (5-10% of initial body weight) lowers the risk for developingobesity-associated diseases such as heart disease and diabetes.

Diabetes mellitus is a chronic condition that is characterized by highblood sugar levels, and insulin resistance. If left untreated, the highblood sugar levels can lead to long-term complications including heartdisease, stroke, diabetic retinopathy, and lower limb amputation.Treatment of diabetes involves controlling and reducing blood sugarlevels and includes exercise and diet modification along withmedications such as insulin and metformin.

One of the approaches used for treating obesity and for glycemic controlinvolves glucagon-like peptide (GLP)-1 receptor agonists that target theincretin pathway. Glucagon-like peptide (GLP)-1 is a peptide hormonesecreted by intestinal enteroendocrine cells. Upon oral glucoseadministration, GLP1 binds to its receptor leading to insulin secretionand a decrease in blood sugar levels (incretin effect). However, GLP1 israpidly inactivated and degraded by the enzyme dipeptidyl peptidase 4(DPP4) and has a very short half-life of 1.5 minutes. Longer-actingderivatives of GLP1 as well as GLP1 receptor agonists including fusionproteins comprising GLP1 have, therefore, been studied for diabetescontrol. GLP1 analogues, fusion proteins and GLP1 receptor agonists aredisclosed, for example, in U.S. Pat. Nos. 7,452,966, 8,389,689,8,497,240, 8,557,769, 8,883,447, 8,895,694, 9,409,966, US20160194371,US20140024586, US20140073563, US20120148586, US20170114115,US20170112904, US20160361390, US20150313908, US20150259416,WO2017074715, WO2016127887, WO2015021871, WO2014113357, EP3034514,EP2470198, and EP2373681.

However, there is a need for novel GLP1 peptide variants and GLP1receptor agonists that are resistant to degradation by DPP4, haveimproved pharmacokinetic properties and have increased potency andsustained in vivo activity in glycemic control. Such GLP1 variants andGLP1 receptor agonists could be used to treat obesity and diabetes.

BRIEF SUMMARY OF THE INVENTION

According to one aspect, the present invention provides glucagon-likepeptide 1 (GLP1) variants comprising at least one amino acidmodification from mature GLP1 (7-37) (SEQ ID NO: 4) selected from thegroup consisting of: (i) addition of an amino acid to the N-terminus;and (ii) deletion of an amino acid from the peptide sequence. In certainembodiments, the modification comprises addition of alanine or glutamineto the N-terminus.

According to one aspect, the present invention provides GLP1 receptoragonists, wherein the GLP1 receptor agonists comprise fusion proteinscomprising GLP1 or a variant thereof. In certain embodiments, the GLP1receptor agonists comprise GLP1 peptide or a GLP1 peptide variant fusedto a stabilizing domain. In one embodiment, the stabilizing domain is anantibody or antigen-binding fragment thereof that binds to GLP1receptor.

The GLP1 receptor agonists of the present invention are useful, interalia, for increasing the binding and/or activity of GLP1. In someembodiments, the GLP1 receptor agonists of the present inventionfunction by activating GLP1 and reducing blood sugar levels. In someembodiments, the GLP1 peptide variants and/or GLP1 receptor agonists ofthe present invention are more resistant to inactivation by dipeptidylpeptidase 4 (DPP4) and show improved half-life in vivo. The improvedGLP1 agonists of the present invention lead to significant reduction inblood sugar levels which is sustained for more than 10 days even with asingle dose. In some embodiments, the GLP1 receptor agonists function bypotentiating glucose-induced secretion of insulin from pancreatic betacells, increasing insulin expression, inhibiting beta-cell apoptosis,promoting beta-cell neogenesis, reducing glucagon secretion, delayinggastric emptying, promoting satiety and increasing peripheral glucosedisposal. In certain embodiments, the GLP1 receptor agonists are usefulin preventing, treating or ameliorating at least one symptom of ahyperglycemia-associated disease or disorder (e.g., diabetes) in asubject. In certain embodiments, the GLP1 receptor agonists may beadministered prophylactically or therapeutically to a subject having orat risk of having diabetes. In certain embodiments, the GLP1 receptoragonists are useful in preventing, treating or ameliorating at least onesymptom or indication of obesity, such as weight loss, in a subject.

In certain embodiments, the GLP1 receptor agonists are fusion proteinscomprising a GLP1 variant and a stabilizing domain, wherein thestabilizing domain comprises an immunoglobulin or fragment thereof. In aspecific embodiment, the immunoglobulin comprises a heavy chain variableregion and a light chain variable region and specifically binds to GLP1receptor. In certain embodiments, the GLP1 receptor agonists bind toGLP1 receptor leading to GLP1 receptor activation. In certainembodiments, the GLP1 receptor agonists function by activating GLP1receptor leading to glycemic control, i.e., reduction of blood glucoselevels.

In one embodiment, the invention provides a fusion protein that has oneor more of the following characteristics: (a) comprises a GLP1 variantdomain and a stabilizing domain; (b) is a GLP1 receptor agonist; (c) theGLP1 variant domain comprises the amino acid sequence of SEQ ID NO: 5,6, 7 or 8; (d) binds to GLP1 receptor; (e) the stabilizing domaincomprises an immunoglobulin or fragment thereof; (f) the stabilizingdomain comprises an immunoglobulin Fc fragment; (g) the stabilizingdomain comprises an anti-GLP1 receptor antibody or antigen-bindingfragment thereof; (h) is resistant to degradation by serum proteases forat least 72 hours; and (i) results in significant reduction in serumglucose level which is sustained for more than 10 days with a singledose of administration.

In one aspect, the present invention provides nucleic acid moleculesencoding GLP1 variants or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the amino acidsequences selected from the group consisting of SEQ ID Nos: 5, 6, 7, 8,9, 10, 11, 12 and 13, or a substantially similar sequence thereof havingat least 90%, at least 95%, at least 98% or at least 99% sequenceidentity thereto.

The present invention also provides nucleic acid molecules encoding anyof the fusion proteins comprising GLP1 variants.

In a related aspect, the present invention provides recombinantexpression vectors capable of expressing a polypeptide comprising a GLP1variant or a fusion protein comprising a GLP1 variant as describedherein. For example, the present invention includes recombinantexpression vectors comprising any of the nucleic acid moleculesmentioned above, i.e., nucleic acid molecules encoding any of the GLP1variants or fusion proteins comprising GLP1 variants. Also includedwithin the scope of the present invention are host cells into which suchvectors have been introduced, as well as methods of producing theproteins or fragments thereof by culturing the host cells underconditions permitting production of the proteins or fragments thereof,and recovering the proteins and fragments so produced.

In one aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of at least onerecombinant protein or fragment thereof which specifically binds GLP1receptor and a pharmaceutically acceptable carrier. In a related aspect,the invention features a composition, which is a combination of a GLP1receptor agonist protein and a second therapeutic agent. In oneembodiment, the second therapeutic agent is any agent that isadvantageously combined with a GLP1 receptor agonist. Exemplary agentsthat may be advantageously combined with a GLP1 receptor agonistinclude, without limitation, other agents that activate GLP1 receptoractivity (including other proteins or metabolites, etc.) and/or agentswhich do not directly bind GLP1 receptor but nonetheless alleviate orameliorate or treat a GLP1 receptor-associated disease or disorder(e.g., diabetes). Additional combination therapies and co-formulationsinvolving the GLP1 receptor agonist proteins of the present inventionare disclosed elsewhere herein.

In another aspect, the invention provides therapeutic methods fortreating a disease or disorder associated with GLP1 such as diabetes ina subject using a GLP1 receptor agonist of the invention, wherein thetherapeutic methods comprise administering a therapeutically effectiveamount of a pharmaceutical composition comprising a GLP1 receptoragonist of the invention to the subject in need thereof. In certainembodiments, the GLP1 receptor agonist comprises a GLP1 variant orfusion protein comprising a GLP1 variant. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by activation of GLP1 receptor activity. In certainembodiments, the invention provides methods to prevent, treat orameliorate at least one symptom of a GLP1 receptor-associated disease ordisorder, the method comprising administering a therapeuticallyeffective amount of a GLP1 receptor agonist of the invention to asubject in need thereof. In some embodiments, the present inventionprovides methods to ameliorate or reduce the severity of at least onesymptom or indication of GLP1 receptor-associated disease or disorder ina subject by administering a therapeutically effective amount of a GLP1receptor agonist protein of the invention, wherein the at least onesymptom or indication is selected from the group consisting of highblood sugar levels, excessive thirst, increased urination, presence ofketones in urine, fatigue, weight fluctuations, blurred vision, slowhealing sores, frequent infections, swollen or tender gums, obesity,heart disease, stroke, kidney disease, eye disease, nerve damage andhigh blood pressure. In certain embodiments, the invention providesmethods to reduce body weight in an overweight or obese subject, themethods comprising administering to the subject a therapeuticallyeffective amount of a GLP1 receptor agonist of the invention that bindsGLP1 receptor and activates GLP1 receptor activity. In certainembodiments, the invention provides methods to reduce blood sugar levelsin a subject, the methods comprising administering to the subject atherapeutically effective amount of a GLP1 receptor agonist of theinvention that binds GLP1 receptor and activates GLP1 receptor activity.In some embodiments, the GLP1 receptor agonist may be administeredprophylactically or therapeutically to a subject having or at risk ofhaving hyperglycemia. The subjects at risk include, but are not limitedto, subjects of advanced age, pregnant women, subjects with high HbA1clevels, and subjects with one or more risk factors including obesity,high blood cholesterol, smoking, excessive alcohol consumption, and/orlack of exercise. In certain embodiments, the invention provides methodsto treat type 2 diabetes that is uncontrolled by treatment with insulinand/or metformin, the methods comprising administering a therapeuticallyeffective amount of a GLP1 receptor agonist of the invention to asubject in need thereof. In certain embodiments, the GLP1 receptoragonist of the invention is administered in combination with a secondtherapeutic agent to the subject in need thereof. The second therapeuticagent may be selected from the group consisting of an insulin or insulinanalogue, a biguanide (e.g., metformin), a thiazolidinedione, asulfonylurea (e.g., chlorpropamide), a glinide (e.g., nateglinide), analpha glucosidase inhibitor, a DPP4 inhibitor (e.g., sitagliptin),pramlintide, bromocriptine, a SGLT2 inhibitor (e.g., canagliflozin), ananti-hypertensive drug, a statin, aspirin, dietary modification,exercise, and a dietary supplement. Additional therapeutic agents thatcan be used in combination with the GLP1 receptor agonist fusionproteins of the present invention are described elsewhere herein. Incertain embodiments, the second therapeutic agent may be an agent thathelps to counteract or reduce any possible side effect(s) associatedwith a GLP1 receptor agonist of the invention, if such side effect(s)should occur. The GLP1 receptor agonist may be administeredsubcutaneously, intravenously, intradermally, intraperitoneally, orally,intramuscularly, or intracranially. The GLP1 receptor agonist may beadministered at a dose of about 0.1 mg/kg of body weight to about 100mg/kg of body weight of the subject. In certain embodiments, a GLP1receptor agonist of the present invention may be administered at one ormore doses comprising between 0.1 mg to 600 mg.

The present invention also includes use of a GLP1 receptor agonist ofthe invention in the manufacture of a medicament for the treatment of adisease or disorder that would benefit from the stimulation of GLP1receptor binding and/or activity (e.g., diabetes including type 2diabetes).

Other embodiments will become apparent from a review of the ensuingdetailed description.

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

The term “GLP1”, also called as “glucagon-like peptide 1”, refers to the31-amino acid peptide hormone released from intestinal L cells followingnutrient consumption. GLP1 binds to GLP1 receptor and potentiates theglucose-induced secretion of insulin from pancreatic beta cells,increases insulin expression, inhibits beta-cell apoptosis, promotesbeta-cell neogenesis, reduces glucagon secretion, delays gastricemptying, promotes satiety and increases peripheral glucose disposal. Incertain embodiments, the term “GLP1” refers to the mature 31 amino acidpeptide hormone (SEQ ID NO: 4) comprising amino acids 7 to 37 of fulllength GLP1 peptide (SEQ ID NO: 3). The term also includes variants ofGLP1 wherein the variants comprise 1, 2, 3, 4, 5 or 6 amino acidsubstitutions, additions or deletions. For example, the term includesvariants that comprise amino acid sequences of SEQ ID NOs: 5, 6, 7, or8.

As used herein, a “stabilizing domain” is any macromolecule that whenfused to a peptide increases the in vivo activity and/or stability ofthe peptide. For example, a stabilizing domain may be a polypeptidecomprising an immunoglobulin C_(H)3 domain. In certain embodiments, thestabilizing domain increases the serum half-life of the peptide. Incertain embodiments, the stabilizing peptide increases the in vivopotency of the peptide. A non-limiting example of a stabilizing domainis an Fc portion of an immunoglobulin, e.g., an Fc domain of an IgGselected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as anyallotype within each isotype group. In certain embodiments, thestabilizing domain is an Fc fragment or an amino acid sequence of 1 toabout 200 amino acids in length containing at least one cysteineresidues. As another example, a stabilizing domain may be animmunoglobulin or an antigen-binding fragment thereof. In certainembodiments, the stabilizing domain is an immunoglobulin comprising aheavy chain variable region and a light chain variable region whereinthe immunoglobulin binds to a specific antigen. In certain embodiments,the stabilizing domain comprises an antigen-binding domain and a Fcdomain (for example, of an IgG1 or IgG4 antibody) or may comprise onlyan antigen-binding portion (for example, a Fab, F(ab′)₂ or scFvfragment), and may be modified to affect functionality. In a specificembodiment, the stabilizing domain is an immunoglobulin comprising aheavy chain variable region and a light chain variable region whereinthe immunoglobulin binds to GLP1 receptor. In other embodiments, thestabilizing domain is a cysteine residue or a short cysteine-containingpeptide. Other stabilizing domains include peptides or polypeptidescomprising or consisting of a leucine zipper, a helix-loop motif, or acoiled-coil motif.

As used herein, the term “GLP1 receptor agonist” refers to a proteinthat binds to GLP1 receptor. In the context of the present invention,the term refers to a fusion protein comprising GLP1 or a GLP1 variantfused to a stabilizing domain. In certain embodiments, the term includesfusion proteins comprising a GLP1 variant fused to an immunoglobulin orfragment thereof. In specific embodiments, the term includes fusionproteins comprising GLP1 or a GLP1 variant fused to the N-terminal oflight chain variable region (VL) of an immunoglobulin. In one specificembodiment, the term includes GLP1 or a GLP1 variant fused to theN-terminal of VL of an antibody or antigen-binding fragment thereof thatbinds to GLP1 receptor.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g. IgM) or antigen-binding fragmentsthereof. Each heavy chain is comprised of a heavy chain constant region(comprised of domains C_(H)1, C_(H)2 and C_(H)3) and an Ig variableregion which may be a heavy chain variable region (“HCVR” or “V_(H)”) ora light chain variable region (“LCVR or “V_(L)”). Each light chain iscomprised of a light chain variable region (“LCVR or “V_(L)”) and alight chain constant region (C_(L)). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, theFRs of the antibody (or antigen binding fragment thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs. The term“antigen-binding protein”, as used herein, also includes antibodies.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Antibodies have been described in the scientificliterature in which one or two CDRs can be dispensed with for binding.Padlan et al. (1995 FASEB J. 9:133-139) analyzed the contact regionsbetween antibodies and their antigens, based on published crystalstructures, and concluded that only about one fifth to one third of CDRresidues actually contact the antigen. Padlan also found many antibodiesin which one or two CDRs had no amino acids in contact with an antigen(see also, Vajdos et al. 2002 J Mol Biol 320:415-428).

Methods and techniques for identifying CDRs within VR amino acidsequences are well known in the art and can be used to identify CDRswithin the specified VR amino acid sequences disclosed herein. Exemplaryconventions that can be used to identify the boundaries of CDRs include,e.g., the Kabat definition, the Chothia definition, and the AbMdefinition. In general terms, the Kabat definition is based on sequencevariability, the Chothia definition is based on the location of thestructural loop regions, and the AbM definition is a compromise betweenthe Kabat and Chothia approaches. See, e.g., Kabat, “Sequences ofProteins of Immunological Interest,” National Institutes of Health,Bethesda, Md. (1991); AI-Lazikani et al., J. Mol. Biol. 273:927-948(1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272(1989). Public databases are also available for identifying CDRsequences within the antigen-binding domain of an antigen-bindingprotein or an antibody.

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in another human antibody sequence or a consensus of suchsequences. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions.

The terms “antigen-binding portion” of an antigen-binding protein,“antigen-binding fragment” of an antigen-binding protein, and the like,as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex. Theterms “antigen-binding fragment” of an antigen-binding protein, or“antigen-binding protein fragment”, as used herein, refers to one ormore fragments of an antigen-binding protein that retain the ability tospecifically bind to GLP1 receptor. An antigen-binding protein fragmentmay include a Fab fragment, a F(ab′)₂ fragment, a Fv fragment, a dAbfragment, a fragment containing a CDR, or an isolated CDR. In certainembodiments, the term “antigen-binding fragment” refers to a polypeptidefragment of a multi-specific antigen-binding molecule. Antigen-bindingfragments of an antigen-binding protein or an antibody may be derived,e.g., from full protein molecules using any suitable standard techniquessuch as proteolytic digestion or recombinant genetic engineeringtechniques involving the manipulation and expression of DNA encodingantigen-binding protein variable and (optionally) constant domains. SuchDNA is known and/or is readily available from, e.g., commercial sources,DNA libraries (including, e.g., phage-antibody libraries), or can besynthesized. The DNA may be sequenced and manipulated chemically or byusing molecular biology techniques, for example, to arrange one or morevariable and/or constant domains into a suitable configuration, or tointroduce codons, create cysteine residues, modify, add or delete aminoacids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antigen-binding protein or an antibodyof the present invention will typically comprise at least oneimmunoglobulin (Ig) variable domain. The variable domain may be of anysize or amino acid composition and will generally comprise at least oneCDR, which is adjacent to or in frame with one or more frameworksequences. In antigen-binding fragments having a V_(H) domain associatedwith a V_(L) domain, the V_(H) and V_(L) domains may be situatedrelative to one another in any suitable arrangement. For example, thevariable region may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantigen-binding protein may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment may contain at leastone variable domain covalently linked to at least one constant domain.Non-limiting, exemplary configurations of variable and constant domainsthat may be found within an antigen-binding fragment of an antibody ofthe present invention include: (i) V_(H)-C_(H)1; (ii) V_(H)-C_(H)2;(iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3, (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antigen-binding protein of the presentinvention may comprise a homo-dimer or hetero-dimer (or other multimer)of any of the variable and constant domain configurations listed abovein non-covalent association with one another and/or with one or moremonomeric V_(H) or V_(L) domain (e.g., by disulfide bond(s)).

As with full protein molecules, antigen-binding fragments may bemono-specific or multi-specific (e.g., bi-specific). A multi-specificantigen-binding fragment of an antigen-binding protein will typicallycomprise at least two different variable domains, wherein each variabledomain is capable of specifically binding to a separate antigen or to adifferent epitope on the same antigen. Any multi-specificantigen-binding protein format, including the exemplary bi-specificantigen-binding protein formats disclosed herein, may be adapted for usein the context of an antigen-binding fragment of an antigen-bindingprotein of the present invention using routine techniques available inthe art.

The terms “fully human antibody”, “human antibody”, “fully humanantigen-binding protein”, or “human antigen-binding protein”, as usedherein, are intended to include antigen-binding proteins having variableand constant regions derived from human germline immunoglobulinsequences. The human antigen-binding proteins of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs and in particular CDR3. However, the term “human antigen-bindingprotein”, as used herein, is not intended to include antigen-bindingproteins in which CDR sequences derived from the germline of anothermammalian species (e.g., mouse), have been grafted onto human FRsequences. The term includes antigen-binding proteins or antibodiesrecombinantly produced in a non-human mammal, or in cells of a non-humanmammal. The term is not intended to include antigen-binding proteins orantibodies isolated from or generated in a human subject.

The term “recombinant”, as used herein, refers to fusion proteins orfragments thereof of the invention created, expressed, isolated orobtained by technologies or methods known in the art as recombinant DNAtechnology which include, e.g., DNA splicing and transgenic expression.The term refers to fusion proteins expressed in a non-human mammal(including transgenic non-human mammals, e.g., transgenic mice), or acell (e.g., CHO cells) expression system or isolated from a recombinantcombinatorial human antibody library.

The term “specifically binds,” or “binds specifically to”, or the like,means that an antibody or antigen-binding fragment thereof forms acomplex with an antigen that is relatively stable under physiologicconditions. Specific binding can be characterized by an equilibriumdissociation constant of at least about 1×10⁻⁸ M or less (e.g., asmaller K_(D) denotes a tighter binding). Methods for determiningwhether two molecules specifically bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,isothermal titration calorimetry, and the like.

The terms “antigen-binding portion” of an antibody, “antigen-bindingfragment” of an antibody, and the like, as used herein, include anynaturally occurring, enzymatically obtainable, synthetic, or geneticallyengineered polypeptide or glycoprotein that specifically binds anantigen to form a complex. The terms “antigen-binding fragment” of anantigen-binding protein or antibody, or “antibody fragment”, as usedherein, refers to one or more fragments of an immunoglobulin proteinthat retain the ability to bind to GLP1 receptor.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular protein-antigeninteraction.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or GAP, as discussed below. A nucleic acid molecule havingsubstantial identity to a reference nucleic acid molecule may, incertain instances, encode a polypeptide having the same or substantiallysimilar amino acid sequence as the polypeptide encoded by the referencenucleic acid molecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 90% sequence identity, even more preferably atleast 95%, 98% or 99% sequence identity. Preferably, residue positions,which are not identical, differ by conservative amino acidsubstitutions. A “conservative amino acid substitution” is one in whichan amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment are wellknown to those of skill in the art. See, e.g., Pearson (1994) MethodsMol. Biol. 24: 307-331, which is herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartate and glutamate, and 7) sulfur-containingside chains: cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443 45, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG Version 6.1. Polypeptide sequences also can be compared usingFASTA with default or recommended parameters; a program in GCG Version6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentsequence identity of the regions of the best overlap between the queryand search sequences (Pearson (2000) supra). Another preferred algorithmwhen comparing a sequence of the invention to a database containing alarge number of sequences from different organisms is the computerprogram BLAST, especially BLASTP or TBLASTN, using default parameters.See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997)Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated byreference.

By the phrase “therapeutically effective amount” is meant an amount thatproduces the desired effect for which it is administered. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see, forexample, Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

As used herein, the term “subject” refers to an animal, preferably amammal, more preferably a human, in need of amelioration, preventionand/or treatment of a disease or disorder associated with GLP1. The termincludes human subjects who have or are at risk of having a disease ordisorder associated with GLP1. For example, the term includes subjectsthat have or are at risk for developing diabetes (e.g., Type 2diabetes). In certain embodiments, the term includes subjects that haveor are at risk for developing obesity, stroke or myocardial infarction.The term also includes subjects that have high blood sugar levels,and/or increased levels of one or more bio markers for diabetes, e.g.,HbA1c. The term also includes subjects with diabetes for whom astandard-of-care therapy (e.g., metformin) is contraindicated or nottolerated or whose disease in uncontrolled despite treatment (e.g., withmetformin).

As used herein, the terms “treat”, “treating”, or “treatment” refer tothe reduction or amelioration of the severity of at least one symptom orindication of a disease or disorder associated with GLP1 due to theadministration of a therapeutic agent such as a GLP1 receptor agonist ofthe present invention to a subject in need thereof. The terms includeinhibition of progression of disease or of worsening of symptoms. Theterms also include positive prognosis of disease, i.e., the subject maybe free of a symptom or indication or may have reduced intensity of asymptom or indication upon administration of a therapeutic agent such asa GLP1 receptor agonist protein of the present invention. For example, asubject with diabetes may have reduction in blood glucose levels uponadministration of a GLP1 receptor agonist of the invention. Thetherapeutic agent may be administered at a therapeutic dose to thesubject.

The terms “prevent”, “preventing” or “prevention” refer to inhibition ofmanifestation of any symptoms or indications of a disease or disorderassociated with hyperglycemia upon administration of a GLP1 receptoragonist of the present invention. The term includes inhibition ofmanifestation of a symptom or indication of a GLP1 receptor-associateddisease or disorder in a subject at risk for developing such a diseaseor disorder.

GLP1 (7-37) (SEQ ID NO: 4) has a very short half-life of in circulation(1-2 minutes), due to its rapid inactivation by the enzyme dipeptidylpeptidase 4 (DPP4). Previous work has shown that various amino acidsubstitutions at position 8 of GLP1 (7-37) make it more resistant toDPP4, thus conferring a longer half-life. However, there is residualsusceptibility of these molecules to DPP4 cleavage (Deacon et al 1998,Diabetologia 41: 271-278). Thus, there is a need to develop newmolecules that have increased resistance to degradation by DPP4.

The present inventors hypothesized that to confer better resistance toDPP4, a first step was to introduce mutations that lengthen or shortenthe amino terminus of GLP1 by either addition of an amino acid (i.e.,Ala, Gln) to the N-terminus or deletion of His or Ala within the peptidesequence to provide better resistance to DPP4 cleavage. The presentinventors have shown herein that these novel GLP1 variants are indeedhighly resistant to degradation by DPP4. A second step was to compensatefor any weakened or reduced GLP1 activity by fusing the GLP1 to ananti-GLP1 receptor antibody, which tethers the weakened GLP1 to the GLP1receptor and thereby increases its potency. Further, the presentinventors found that these fusion proteins, likely because they containan Fc domain, had an increased serum half-life and led to increasedreduction of blood sugar levels that was sustained for more than 10days. The novel GLP1 variants and fusion proteins disclosed herein havesignificantly improved resistance to degradation by DPP4 in vitro and invivo and show vastly improved potency in glycemic control as shownherein. The terms “significantly improved” or “enhanced” or “increased”,as used herein, in the context of resistance to degradation by DPP4refer to increased resistance to degradation upon incubation with DPP4for more than 4 hours, more than 8 hours, more than 16 hours, more than24 hours, more than 36 hours or more than 70 hours, as measured by theassays described herein. The terms “significantly improved” or“enhanced” or “increased”, as used herein, in the context of reductionof blood sugar levels refers to sustained lowering of blood sugar levelsfor more than 1 day, more than 2 days, more than 3 days, more than 4days, more than 5 days, more than 6 days, more than 7 days, more than 8days, more than 9 days, or more than 10 days in a subject uponadministration of a GLP1 receptor agonist of the present invention.

The GLP1 receptor agonists of the present invention bind to GLP1receptor with high affinity and lead to GLP1 receptor activation. Insome embodiments, the proteins are useful for treating a subjectsuffering from diabetes. The proteins when administered to a subject inneed thereof may reduce blood sugar levels in the subject. They may beused alone or as adjunct therapy with other therapeutic moieties ormodalities known in the art for treating hyperglycemia.

Certain GLP1 receptor agonist proteins of the present invention are ableto bind to and stimulate the activity of GLP1 receptor, as determined byin vitro or in vivo assays. The ability of the proteins of the inventionto bind to and enhance the activity of GLP1 receptor may be measuredusing any standard method known to those skilled in the art, includingbinding assays, or activity assays, as described herein.

The antigen-binding proteins specific for GLP1 receptor may contain noadditional labels or moieties, or they may contain an N-terminal orC-terminal label or moiety. In one embodiment, the label or moiety isbiotin. In a binding assay, the location of a label (if any) maydetermine the orientation of the peptide relative to the surface uponwhich the peptide is bound. For example, if a surface is coated withavidin, a peptide containing an N-terminal biotin will be oriented suchthat the C-terminal portion of the peptide will be distal to thesurface. In one embodiment, the label may be a radionuclide, afluorescent dye or a MRI-detectable label. In certain embodiments, suchlabeled antigen-binding proteins may be used in diagnostic assaysincluding imaging assays.

Bioequivalents

The GLP1 receptor agonists of the present invention encompass proteinshaving amino acid sequences that vary from those of the described GLP1receptor agonists, but that retain the ability to bind GLP1 receptor.Such variant GLP1 receptor agonists comprise one or more additions,deletions, or substitutions of amino acids when compared to parentsequence, but exhibit biological activity that is essentially equivalentto that of the described GLP1 receptor agonists. Likewise, the GLP1receptor agonist-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode a GLP1 receptor agonist that is essentiallybioequivalent to a GLP1 receptor agonist of the invention.

Two proteins are considered bioequivalent if, for example, they arepharmaceutical equivalents or pharmaceutical alternatives whose rate andextent of absorption do not show a significant difference whenadministered at the same molar dose under similar experimentalconditions, either single dose or multiple doses. Some proteins will beconsidered equivalents or pharmaceutical alternatives if they areequivalent in the extent of their absorption but not in their rate ofabsorption and yet may be considered bioequivalent because suchdifferences in the rate of absorption are intentional and are reflectedin the labeling, are not essential to the attainment of effective bodydrug concentrations on, e.g., chronic use, and are considered medicallyinsignificant for the particular drug product studied.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalentif there are no clinically meaningful differences in their safety,purity, or potency.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalentif a patient can be switched one or more times between the referenceproduct and the biological product without an expected increase in therisk of adverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two GLP1 receptor agonist proteins are bioequivalentif they both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and/or in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the protein or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the protein (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antigen-binding protein.

Bioequivalent variants of the GLP1 receptor agonist proteins of theinvention may be constructed by, for example, making varioussubstitutions of residues or sequences or deleting terminal or internalresidues or sequences not needed for biological activity. For example,cysteine residues not essential for biological activity can be deletedor replaced with other amino acids to prevent formation of unnecessaryor incorrect intramolecular disulfide bridges upon renaturation. Inother contexts, bioequivalent proteins may include variants comprisingamino acid changes, which modify the glycosylation characteristics ofthe proteins, e.g., mutations that eliminate or remove glycosylation.

Biological Characteristics of the GLP1 Receptor Agonists

In general, the GLP1 receptor agonists of the present invention functionby binding to GLP1 receptor and facilitate activation of GLP1 receptorupon binding. In certain embodiments, the proteins of the presentinvention bind with high affinity to GLP1 receptor. For example, thepresent invention includes GLP1 receptor agonists that that lead toactivation of the GLP1 receptor (e.g., at 25° C. or at 37° C.) asmeasured by luciferase assay, e.g., using the assay format as defined inExample 2 herein. In certain embodiments, the GLP1 receptor agonistsactivate GLP1 receptor with a EC50 of less than 10 nM, less than 500 pM,or less than 250 pM, as measured by luciferase assay, e.g., using theassay format as defined in Example 2 herein, or a substantially similarassay.

The present invention also includes GLP1 receptor agonists that reduceblood sugar levels in vivo upon administration to a subject in needthereof, e.g., as shown in Example 3, or a substantially similar assay.The GLP1 receptor agonists affect enhanced glycemic control uponadministration, leading to reduction in blood glucose levels. In certainembodiments, even a single therapeutically effective dose of the GLP1receptor agonists of the present invention leads to significant bloodsugar reduction, which is sustained for more than 10 days.

The present invention also includes GLP1 receptor agonists that showenhanced resistance to degradation by serum proteases/peptidases, asmeasured by mass spectroscopy, e.g., as shown in Example 4 herein, or asubstantially similar method. In certain embodiments, the GLP1 receptoragonists are resistant to degradation by dipeptidyl peptidase 4 (DPP4)for more than 4 hours, more than 6 hours, more than 12 hours, more than24 hours, more than 48 hours, or more than 70 hours, as measured by theassay described in Example 4 herein.

The GLP1 receptor agonists of the present invention may possess one ormore of the aforementioned biological characteristics, or anycombinations thereof. Other biological characteristics of the proteinsof the present invention will be evident to a person of ordinary skillin the art from a review of the present disclosure including the workingExamples herein.

Therapeutic Administration and Formulations

The invention provides therapeutic compositions comprising the GLP1receptor agonists of the present invention. Therapeutic compositions inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

The dose of GLP1 receptor agonist may vary depending upon the age andthe size of a subject to be administered, target disease, conditions,route of administration, and the like. When an antigen-binding proteinof the present invention is used for treating a disease or disorder inan adult patient, or for preventing such a disease, it is advantageousto administer the antigen-binding protein of the present inventionnormally at a single dose of about 0.001 to about 100 mg/kg body weight,more preferably about 0.001 to about 60, about 0.01 to about 10, orabout 0.01 to about 1 mg/kg body weight. Depending on the severity ofthe condition, the frequency and the duration of the treatment can beadjusted. In certain embodiments, the antigen-binding protein orantigen-binding fragment thereof of the invention can be administered asan initial dose of at least about 0.001 mg to about 100 mg, about 0.001to about 50 mg, about 0.005 to about 50 mg, about 0.01 to about 40 mg,to about 30 mg, or to about 10 mg. In certain embodiments, the initialdose may be followed by administration of a second or a plurality ofsubsequent doses of the GLP1 receptor agonist in an amount that can beapproximately the same or less than that of the initial dose, whereinthe subsequent doses are separated by at least 1 day to 3 days; at leastone week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, transdermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. The pharmaceutical composition can be also deliveredin a vesicle, in particular a liposome (see, for example, Langer (1990)Science 249:1527-1533).

The use of nanoparticles to deliver the GLP1 receptor agonists of thepresent invention is also contemplated herein. Protein-conjugatednanoparticles may be used both for therapeutic and diagnosticapplications. Nanoparticles may be developed and conjugated toantigen-binding proteins contained in pharmaceutical compositions totarget cells. Nanoparticles for drug delivery have also been describedin, for example, U.S. Pat. No. 8,257,740, or U.S. Pat. No. 8,246,995,each incorporated herein in its entirety.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used.In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous, intracranial, intraperitoneal andintramuscular injections, drip infusions, etc. These injectablepreparations may be prepared by methods publicly known. For example, theinjectable preparations may be prepared, e.g., by dissolving, suspendingor emulsifying the antigen-binding protein or its salt described abovein a sterile aqueous medium or an oily medium conventionally used forinjections. As the aqueous medium for injections, there are, forexample, physiological saline, an isotonic solution containing glucoseand other auxiliary agents, etc., which may be used in combination withan appropriate solubilizing agent such as an alcohol (e.g., ethanol), apolyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionicsurfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)], etc. As the oily medium, there areemployed, e.g., sesame oil, soybean oil, etc., which may be used incombination with a solubilizing agent such as benzyl benzoate, benzylalcohol, etc. The injection thus prepared is preferably filled in anappropriate ampoule.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier,Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (AbbottLabs, Abbott Park, Ill.), to name only a few.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the GLP1 receptor agonistcontained is generally about 0.001 to about 100 mg per dosage form in aunit dose; especially in the form of injection, it is preferred that theGLP1 receptor agonist is contained in about 0.001 to about 100 mg and inabout 0.01 to about 100 mg for the other dosage forms.

Therapeutic Uses of the GLP1 Receptor Agonists

The GLP1 receptor agonists of the present invention are useful for thetreatment, and/or prevention of a disease or disorder or conditionassociated with hyperglycemia such as diabetes and/or for amelioratingat least one symptom associated with such disease, disorder orcondition. In one embodiment, a GLP1 receptor agonist of the inventionmay be administered at a therapeutic dose to a patient with diabetes(e.g., type 2 diabetes).

In certain embodiments, the GLP1 receptor agonists of the invention areuseful to treat subjects suffering from a disease or disorder selectedfrom the group consisting of diabetes mellitus, obesity, insulinresistance, hypertension, dyslipidemia, Type 2 diabetes, Type 1diabetes, pre-diabetes, cardiovascular disease, atherosclerosis,congestive heart failure, coronary heart disease, arteriosclerosis,peripheral artery disease, stroke, respiratory dysfunction, renaldisease, fatty liver disease, non-alcoholic steatohepatitis (NASH), andmetabolic syndrome.

In certain embodiments, the GLP1 receptor agonists of the invention areuseful to treat subjects that are overweight or obese and/or prevent ortreat one or more obesity-associated disorders such as heart disease,stroke, and diabetes.

In certain embodiments, the GLP1 receptor agonists of the invention areuseful to treat subjects suffering from diabetes and/or prevent one ormore complications of diabetes such as heart disease, stroke, kidneydisease, retinopathy, blindness and nerve damage.

It is also contemplated herein to use one or more GLP1 receptor agonistproteins of the present invention prophylactically to subjects at riskfor developing diabetes (e.g., type 2 diabetes). The subjects at riskinclude, but are not limited to, subjects of advanced age, pregnantwomen, and subjects with one or more risk factors including familyhistory of obesity, high blood cholesterol, smoking, excessive alcoholconsumption, and/or lack of exercise.

In a further embodiment, the proteins of the invention are used for thepreparation of a pharmaceutical composition or medicament for treatingpatients suffering from a disease or disorder such as diabetes andobesity. In another embodiment of the invention, the present GLP1receptor agonists are used as adjunct therapy with any other agent orany other therapy known to those skilled in the art useful for treatingor ameliorating a disease or disorder associated with hyperglycemia suchas diabetes (e.g., type 2 diabetes).

Combination Therapies

Combination therapies may include a GLP1 receptor agonist of theinvention and any additional therapeutic agent that may beadvantageously combined with the GLP1 receptor agonist of the invention,or with a biologically active fragment thereof of the invention. TheGLP1 receptor agonists of the present invention may be combinedsynergistically with one or more drugs or therapy used to treat anydisease or disorder associated with hyperglycemia (e.g., diabetes). Insome embodiments, the GLP1 receptor agonists of the invention may becombined with a second therapeutic agent to reduce blood sugar levels ina subject, or to ameliorate one or more symptoms of diabetes.

The GLP1 receptor agonists of the present invention may be used incombination with an insulin (insulin or an insulin analog), insulinsensitizers such as biguanides (e.g., metformin), and thiazolidinediones(e.g., rosiglitazone), insulin secretagogues such as sulphonylureas(e.g., chlorpropamide), and glinides (e.g., nateglinide),alpha-glucosidase inhibitors (e.g., acarbose), dipeptidyl peptidase 4(DPP4) inhibitors (e.g., sitagliptin), pramlinitide, bromocriptine,sodium glucose cotransporter 2 (SGLT-2) inhibitors (e.g.,canagliflozin), an anti-hypertensive drug (e.g., anangiotensin-converting enzyme inhibitor, an angiotensin receptorblocker, a diuretic, a calcium channel blocker, an alpha-adrenoceptorblocker, an endothelin-1 receptor blocker, an organic nitrate, and aprotein kinase C inhibitor), a statin, aspirin, a different GLP1receptor agonist, a dietary supplement or any other therapy (e.g.,exercise) to treat or manage diabetes. In certain embodiments, the GLP1receptor agonists of the present invention may be administered incombination with a second therapeutic agent or therapy selected from thegroup consisting of insulin, an insulin analog, metformin,rosiglitazone, pioglitazone, chlorpropamide, glibenclamide, glimepiride,glipizide, tolazamide, tolbutamide, nateglinide, repaglinide, acarbose,miglitol, exenatide, liraglutide, albiglutide, dulaglutide, sitagliptin,saxagliptin, linagliptin, alogliptin, pramlinitide, bromocriptinequick-release, canagliflozin, dapagliflozin, empagliflozin, dietmodifications and exercise.

As used herein, the term “in combination with” means that additionaltherapeutically active component(s) may be administered prior to,concurrent with, or after the administration of the GLP1 receptoragonist of the present invention. The term “in combination with” alsoincludes sequential or concomitant administration of a GLP1 receptoragonist and a second therapeutic agent.

The additional therapeutically active component(s) may be administeredto a subject prior to administration of a GLP1 receptor agonist of thepresent invention. For example, a first component may be deemed to beadministered “prior to” a second component if the first component isadministered 1 week before, 72 hours before, 60 hours before, 48 hoursbefore, 36 hours before, 24 hours before, 12 hours before, 6 hoursbefore, 5 hours before, 4 hours before, 3 hours before, 2 hours before,1 hour before, 30 minutes before, 15 minutes before, 10 minutes before,5 minutes before, or less than 1 minute before administration of thesecond component. In other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject afteradministration of a GLP1 receptor agonist of the present invention. Forexample, a first component may be deemed to be administered “after” asecond component if the first component is administered 1 minute after,5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after,6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hoursafter, 60 hours after, 72 hours after administration of the secondcomponent. In yet other embodiments, the additional therapeuticallyactive component(s) may be administered to a subject concurrent withadministration of a GLP1 receptor agonist of the present invention.“Concurrent” administration, for purposes of the present invention,includes, e.g., administration of a GLP1 receptor agonist and anadditional therapeutically active component to a subject in a singledosage form, or in separate dosage forms administered to the subjectwithin about 30 minutes or less of each other. If administered inseparate dosage forms, each dosage form may be administered via the sameroute (e.g., both the GLP1 receptor agonist and the additionaltherapeutically active component may be administered intravenously,etc.); alternatively, each dosage form may be administered via adifferent route (e.g., the GLP1 receptor agonist may be administeredintravenously, and the additional therapeutically active component maybe administered orally). In any event, administering the components in asingle dosage from, in separate dosage forms by the same route, or inseparate dosage forms by different routes are all considered “concurrentadministration,” for purposes of the present disclosure. For purposes ofthe present disclosure, administration of a GLP1 receptor agonist “priorto”, “concurrent with,” or “after” (as those terms are defined hereinabove) administration of an additional therapeutically active componentis considered administration of a GLP1 receptor agonist “in combinationwith” an additional therapeutically active component.

The present invention includes pharmaceutical compositions in which aGLP1 receptor agonist of the present invention is co-formulated with oneor more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments, a single dose of a GLP1 receptoragonist of the invention (or a pharmaceutical composition comprising acombination of a GLP1 receptor agonist and any of the additionaltherapeutically active agents mentioned herein) may be administered to asubject in need thereof. According to certain embodiments of the presentinvention, multiple doses of a GLP1 receptor agonist (or apharmaceutical composition comprising a combination of a GLP1 receptoragonist and any of the additional therapeutically active agentsmentioned herein) may be administered to a subject over a defined timecourse. The methods according to this aspect of the invention comprisesequentially administering to a subject multiple doses of a GLP1receptor agonist of the invention. As used herein, “sequentiallyadministering” means that each dose of GLP1 receptor agonist isadministered to the subject at a different point in time, e.g., ondifferent days separated by a predetermined interval (e.g., hours, days,weeks or months). The present invention includes methods which comprisesequentially administering to the patient a single initial dose of aGLP1 receptor agonist, followed by one or more secondary doses of theGLP1 receptor agonist, and optionally followed by one or more tertiarydoses of the GLP1 receptor agonist.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the GLP1 receptor agonistof the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of GLP1receptor agonist, but generally may differ from one another in terms offrequency of administration. In certain embodiments, however, the amountof GLP1 receptor agonist contained in the initial, secondary and/ortertiary doses varies from one another (e.g., adjusted up or down asappropriate) during the course of treatment. In certain embodiments, oneor more (e.g., 2, 3, 4, or 5) doses are administered at the beginning ofthe treatment regimen as “loading doses” followed by subsequent dosesthat are administered on a less frequent basis (e.g., “maintenancedoses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 48 hours (e.g., 1,1%, 2, 2%, 3, 3%, 4, 4%, 5, 5%, 6, 6%, 7, 7%, 8, 8%, 9, 9%, 10, 10%, 11,11%, 12, 12%, 13, 13%, 14, 14%, 15, 15%, 16, 16%, 17, 17%, 18, 18%, 19,19%, 20, 20%, 21, 21%, 22, 22%, 23, 23%, 24, 24%, 25, 25%, 26, 26%, ormore) after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of the GLP1 receptor agonist which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses. In certain embodiments,each secondary and/or tertiary dose is administered every day, every 2days, 3 days, 4 days, 5 days, 6 days, or 7 days after the immediatelypreceding dose. In certain embodiments, each secondary and/or tertiarydose is administered every 0.5 weeks, 1 week, 2 weeks, 3 weeks, or 4weeks after the immediately preceding dose.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof a GLP1 receptor agonist. For example, in certain embodiments, only asingle secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In certain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

Dosage

The amount of GLP1 receptor agonist administered to a subject accordingto the methods of the present invention is, generally, a therapeuticallyeffective amount. As used herein, the phrase “therapeutically effectiveamount” means an amount of GLP1 receptor agonist that results in one ormore of: (a) reduction of high sugar levels to normal levels (e.g.,pre-prandial blood glucose levels of 80-130 mg/dL; and/or (b) adetectable improvement in one or more symptoms or indicia of diabetes.

In the case of a GLP1 receptor agonist, a therapeutically effectiveamount can be from about 0.001 mg to about 100 mg, e.g., about 0.001 mg,about 0.002 mg, about 0.003 mg, about 0.004 mg, about 0.005 mg, about0.006 mg, about 0.007 mg, about 0.008 mg, about 0.009 mg, about 0.01 mg,about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg,about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg,about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg,about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about90 mg, about 95 mg, or about 100 mg of the GLP1 receptor agonist. Incertain embodiments, 0.005 mg to 50 mg, 0.005 mg to 30 mg, 0.005 mg to10 mg, 0.1 mg to 10 mg, or 0.1 mg to 5 mg of a GLP1 receptor agonist isadministered to a subject in need thereof.

The amount of GLP1 receptor agonist contained within the individualdoses may be expressed in terms of milligrams of antibody per kilogramof subject body weight (i.e., mg/kg). For example, the GLP1 receptoragonist may be administered to a subject at a dose of about 0.0001 toabout 100 mg/kg of subject body weight.

Selected Embodiments

In Embodiment, 1, the present invention provides a glucagon-like peptide1 (GLP1) variant comprising mature GLP1 (7-37) (SEQ ID NO: 4) having atleast one amino acid modification selected from the group consisting of:(i) addition of an amino acid to the N-terminus; and (ii) deletion of anamino acid from the peptide sequence; wherein the GLP1 variant hasenhanced resistance to proteolytic cleavage and/or enhanced bloodglucose lowering ability.

In Embodiment, 2, the present invention provides the GLP1 variant ofembodiment 1, wherein the amino acid modification comprises addition ofan amino acid selected from the group consisting of alanine (Ala) andglutamine (Gln) to the N-terminus.

In Embodiment 3, the present invention provides the GLP1 variant ofembodiment 1 or 2, wherein the amino acid modification comprisesaddition of Gln to the N-terminus.

In Embodiment 4, the present invention provides the GLP1 variant ofembodiment 1, wherein the amino acid modification comprises deletion ofhistidine (His1) or alanine (Ala2) from SEQ ID NO: 4.

In Embodiment 5, the present invention provides the GLP1 variant of anyone of embodiment 1-4 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 5, 6, 7 and 8.

In Embodiment 6, the present invention provides the GLP1 variant ofembodiment 5 comprising an amino acid sequence of SEQ ID NO: 6.

In Embodiment 7, the present invention provides a fusion proteincomprising the GLP1 variant of any one of embodiments 1-6 fused to astabilizing domain, wherein the stabilizing domain is an antigen-bindingprotein or antigen-binding fragment thereof that specifically binds GLP1receptor and that comprises a heavy chain variable region (HCVR) and alight chain variable region (LCVR).

In Embodiment 8, the present invention provides the fusion protein ofembodiment 7, wherein the GLP1 variant is fused to the N-terminal orC-terminal of the HCVR of the antigen-binding protein or antigen-bindingfragment thereof.

In Embodiment 9, the present invention provides the fusion protein ofembodiment 7, wherein the GLP1 variant is fused to the N-terminal orC-terminal of the LCVR of the antigen-binding protein or antigen-bindingfragment thereof.

In Embodiment 10, the present invention provides a fusion proteincomprising a GLP1 variant of any one of embodiments 1-6 fused to astabilizing domain, wherein the stabilizing domain is an immunoglobulin(Ig) or fragment thereof.

In Embodiment 11, the present invention provides the fusion protein ofembodiment 11 comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos: 9, 10, 11, 12 and 13.

In Embodiment 12, the present invention provides a fusion proteincomprising a GLP1 variant fused to a stabilizing domain, wherein thestabilizing domain is an antigen-binding protein or antigen-bindingfragment thereof, wherein the antigen-binding protein or fragmentthereof comprises a heavy chain variable region (HCVR) and a light chainvariable region (LCVR).

In Embodiment 13, the present invention provides the fusion protein ofembodiment 12, wherein the GLP1 variant is fused to N-terminal orC-terminal of the HCVR of the antigen-binding protein or fragmentthereof.

In Embodiment 14, the present invention provides the fusion protein ofembodiment 12, wherein the GLP1 variant is fused to N-terminal orC-terminal of the LCVR of the antigen-binding protein or fragmentthereof.

In Embodiment 15, the present invention provides the fusion protein ofany one of embodiments 12-14, wherein the antigen-binding protein orfragment thereof binds specifically to GLP1 receptor.

In Embodiment 16, the present invention provides the fusion protein ofany one of embodiments 12-15 comprising the GLP1 variant of any of theabove embodiments.

In Embodiment 17, the present invention provides the fusion protein ofany one of embodiments 12-16, wherein the GLP1 variant comprises anamino acid sequence selected from the group consisting of SEQ ID NOs: 5,6, 7 and 8.

In Embodiment 18, the present invention provides the fusion protein ofembodiment 16 or 17, wherein the GLP1 variant comprises an amino acidsequence of SEQ ID NO: 6.

In Embodiment 19, the present invention provides a GLP1 receptor agonistcomprising a GLP1 variant wherein the GLP1 variant is fused to astabilizing domain, wherein the stabilizing domain is an antigen-bindingprotein or antigen-binding fragment thereof, wherein the antigen-bindingprotein or fragment thereof comprises a heavy chain variable region(HCVR) and a light chain variable region (LCVR).

In Embodiment 20, the present invention provides the GLP1 receptoragonist of embodiment 19, wherein the GLP1 variant is fused toN-terminal or C-terminal of the HCVR of the antigen-binding protein orfragment thereof.

In Embodiment 21, the present invention provides the GLP1 receptoragonist of embodiment 19, wherein the GLP1 variant is fused toN-terminal or C-terminal of the LCVR of the antigen-binding protein orfragment thereof.

In Embodiment 22, the present invention provides the GLP1 receptoragonist of any one of embodiments 19-21, wherein the antigen-bindingprotein or fragment thereof binds specifically to GLP1 receptor.

In Embodiment 23, the present invention provides the GLP1 receptoragonist of any one of embodiments 19-22 comprising the GLP1 variant ofembodiment 1.

In Embodiment 24, the present invention provides the GLP1 receptoragonist of any one of embodiments 19-23, wherein the GLP1 variantcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 6, 7 and 8.

In Embodiment 25, the present invention provides the GLP1 receptoragonist of embodiment 23 or 24, wherein the GLP1 variant comprises anamino acid sequence of SEQ ID NO: 6.

In Embodiment 26, the present invention provides a GLP1 receptor agonistcomprising a GLP1 variant wherein the GLP1 variant is fused to astabilizing domain, wherein the stabilizing domain is an immunoglobulin(Ig) or fragment thereof.

In Embodiment 27, the present invention provides the GLP1 receptoragonist of embodiment 26 comprising an amino acid sequence selected fromthe group consisting of SEQ ID Nos: 9, 10, 11, 12 and 13.

In Embodiment 28, the present invention provides a pharmaceuticalcomposition comprising a protein of any one of embodiments 1-27 and apharmaceutically acceptable carrier or diluent.

In Embodiment 29, the present invention provides an isolatedpolynucleotide molecule comprising a polynucleotide sequence thatencodes a GLP1 variant as set forth in any one of embodiments 1-6.

In Embodiment 30, the present invention provides an isolatedpolynucleotide molecule comprising a polynucleotide sequence thatencodes a fusion protein as set forth in any one of embodiments 10-11.

In Embodiment 31, the present invention provides an isolatedpolynucleotide molecule comprising a polynucleotide sequence thatencodes a GLP1 receptor agonist as set forth in any one of embodiments26-27.

In Embodiment 32, the present invention provides a vector comprising thepolynucleotide sequence of any one of embodiments 29-31.

In Embodiment 33, the present invention provides a cell expressing thevector of embodiment 32.

In Embodiment 34, the present invention provides a method of loweringblood sugar level comprising administering a pharmaceutical compositioncomprising a therapeutically effective amount of the protein of any oneof embodiments 1-27 to a subject in need thereof.

In Embodiment 35, the present invention provides the method ofembodiment 34, wherein the subject has a disease or disorder selectedfrom the group consisting of diabetes mellitus, obesity, insulinresistance, hypertension, dyslipidemia, Type 2 diabetes, Type 1diabetes, pre-diabetes, cardiovascular disease, atherosclerosis,congestive heart failure, coronary heart disease, arteriosclerosis,peripheral artery disease, stroke, respiratory dysfunction, renaldisease, fatty liver disease, non-alcoholic steatohepatitis (NASH), andmetabolic syndrome.

In Embodiment 36, the present invention provides a method of preventing,treating or ameliorating at least one symptom, indication orcomplication of Type 2 diabetes, the method comprising administering apharmaceutical composition comprising a therapeutically effective amountof the protein of any one of embodiments 1-27 to a subject in needthereof.

In Embodiment 37, the present invention provides the method ofembodiment 36, wherein the at least one symptom, indication orcomplication is selected from the group consisting of high blood sugarlevels, excessive thirst, increased urination, presence of ketones inurine, fatigue, weight fluctuations, blurred vision, slow healing sores,frequent infections, swollen or tender gums, obesity, heart disease,stroke, kidney disease, eye disease, nerve damage and high bloodpressure.

In Embodiment 38, the present invention provides the method of any oneof embodiments 34-37, wherein the pharmaceutical composition isadministered in combination with a second therapeutic agent or therapy.

In Embodiment 39, the present invention provides the method ofembodiment 38, wherein the second therapeutic agent or therapy isselected from the group consisting of an insulin or insulin analogue, abiguanide (e.g., metformin), a thiazolidinedione, a sulfonylurea (e.g.,chlorpropamide), a glinide (e.g., nateglinide), an alpha glucosidaseinhibitor, a DPP4 inhibitor (e.g., sitagliptin), pramlintide,bromocriptine, a SGLT2 inhibitor (e.g., canagliflozin), ananti-hypertensive drug, a statin, aspirin, dietary modification,exercise, and a dietary supplement.

In Embodiment 40, the present invention provides the method of any oneof embodiments 34-39, wherein the pharmaceutical composition isadministered subcutaneously, intravenously, intradermally,intraperitoneally, orally, or intramuscularly.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, room temperatureis about 25° C., and pressure is at or near atmospheric.

Example 1: Exemplary Fusion Proteins Comprising GLP1

GLP1 (7-37) has a very short half-life of in circulation (1-2 minutes),due to its rapid inactivation by the enzyme dipeptidyl peptidase 4(DPP4). Previous work has shown that various amino acid substitutions atposition 8 of GLP1 (7-37) make it more resistant to DPP4, thusconferring a longer half-life (Deacon et al 1998, Diabetologia 41:271-278). However, there is residual susceptibility of these moleculesto DPP4 cleavage. Thus there is a need to develop new molecules that arestill more resistant to DPP4.

To confer better resistance to DPP4, a first part of the technology isto introduce mutations that lengthen or shorten the amino terminus ofGLP1 by either addition of an amino acid (i.e., Ala, Gln) to theN-terminus or deletion of His7 or Ala8 within the peptide sequence toprovide better resistance to DPP4 cleavage. These modifications alsoweaken the GLP1 activity, and a second part of the technology is tocompensate for the reduced activity by tethering the weakened agonist tothe receptor using an anti GLP1R antibody by fusing the peptide to theN-terminus of the anti-GLP1R antibody light chain sequence. As proof ofconcept, modified GLP1 (7-37) ligand sequences were fused to theN-terminus of the light chain of a GLP1R antibody, as described below.

Mature GLP1 is a 31-amino acid peptide hormone comprising amino acids 7to 37 of full-length GLP1 (SEQ ID NO: 3) and has the amino acid sequenceHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 4).

Mature GLP1 was modified by amino acid deletions or additions at theamino terminus to generate GLP1 variants. Exemplary GLP1 variants aregiven below:

Des-Ala-GLP1 comprising the amino acid sequenceHEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 5)

-   -   Q-GLP1 comprising the amino acid sequence        QHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 6)    -   A-GLP1 comprising the amino acid sequence        AHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 7)    -   desH-GLP1 comprising the sequence AEGTFTSDVSSYLEGQAAKEFIAWLVKGRG        (SEQ ID NO: 8)

To compensate for possible reduced activity of the above GLP1 variants,they were tethered to the GLP1 receptor (GLP1R) using an anti-GLP1Rantibody or to an antibody Fc fragment. Exemplary fusion proteinscomprising mature GLP1 or a GLP1 variant were generated by fusing matureGLP1 or the GLP1 variant to the N-terminal of the light chain of ananti-GLP1 receptor antibody comprising heavy chain variable region ofSEQ ID NO: 1 and light chain variable region (LCVR) of SEQ ID NO: 2(hereinafter referred to as “mAb1”; US Patent Application PublicationNo. 20060275288 [Abbott Laboratories]) and are listed below:

-   -   Des-Ala-GLP1-mAb1: Des-Ala-GLP1 (SEQ ID NO: 5) fused to        N-terminus of light chain of mAb1    -   Q-GLP1-mAb1: Q-GLP1 (SEQ ID NO: 6) fused to N-terminus of light        chain of mAb1    -   A-GLP1-mAb1: A-GLP1 (SEQ ID NO: 7) fused to N-terminus of light        chain of mAb1

Fusion proteins comprising mature GLP1 or GLP1 variant and animmunoglobulin Fc fragment were also generated and are listed below:

-   -   GLP1-hFc (SEQ ID NO: 9)    -   A-GLP1-hFc (SEQ ID NO: 10)    -   Q-GLP1-hFc (SEQ ID NO: 11)    -   Des-Ala-GLP1-hFc (SEQ ID NO: 12)    -   desH-GLP1-hFc (SEQ ID NO: 13)        Control Construct        Comparator: A GLP1 analogue having the amino acid sequence        characteristics of LY2189265 fused to hIgG4 Fc domain        (dulaglutide; Eli Lilly), as disclosed in Glaesner et al 2010        (Diabetes Metab. Res. Rev. 26: 287-296) was used as a comparator        (SEQ ID NO: 14) in the following Examples.

Example 2: Luciferase Assay

The GLP1 fusion proteins were tested for their ability to stimulate cAMPproduction in a reporter cell line 293/FSC11/Cre-Luc that stablyexpresses the human GLP1 receptor together with a luciferase codingsequence under the control of a cre promoter that responds to cAMP.

For the luciferase bioassay, the 293/FSC11/Cre-Luc GLP1R stable cellswere seeded into 96-well assay plates at 30,000 cells/well in OPTIMEMsupplemented with 0.1% FBS and then incubated at 37° C. in 5% CO₂overnight. The next day, to determine the dose response of testproteins, human GLP1 (Phoenix #028-13), des-Ala-GLP1-mAb1, Q-GLP1-mAb1,or A-GLP1-mAb1 were tested in the assay. All test compounds werepurified proteins, except A-GLP1-mAb1 which was used directly fromculture media after transient transfection of CHO cells with a vectorencoding the modified antibody. The material in culture media wasquantitated by ELISA. Test samples were added to cells at concentrationsranging from 0.02 pM to 100 nM.

After 5.5 hours or overnight incubation at 37° C. in 5% CO₂, OneGloreagent (Promega, #E6051) was added to the samples and luciferaseactivity was then measured using a Victor X (Perkin Elmer) plate reader.The results were analyzed using nonlinear regression (three parameter)with Prism 6 software (GraphPad) to obtain EC₅₀ values.

As shown in the Table 1, the Q- and A-modified mAb1 antibody fusionsshow EC50 values of 204 pM and 312 pM, respectively, for GLP1Ractivation.

TABLE 1 EC50 for GLP1 fusion proteins GLP1 fusion proteins EC50des-Ala-GLP1-mAbl 10 nM Q-GLP1-mAb1 0.204 nM A-GLP1-mAb1 0.312 nMGLP1-hFc 0.147 nM A-GLP1-hFc 120 nM Q-GLP1-hFc 135 nM Des-Ala-GLP1-hFcNot detectable Des-H-GLP1-hFc 2560 nM Comparator 0.059 nM

The EC50 for des-Ala-GLP1-mAb1 was 10 nM. The EC50 for Q- and A-GLP1-hFcis only 135 nM and 120 nM, while the EC50 for Des A-GLP1-hFc isundetectable.

Example 3: Effect of Q-GLP1 Fused to GLP1R Antibody on Blood Glucose andGlucose Tolerance in GLP1R Humanized Mice

The effect of Q-GLP1 fused to the N-terminus of the light chain of ananti-GLP1R antibody (Q-GLP1-mAb1) on blood glucose and glucose tolerancewas determined in genetically engineered mice expressing the human GLP1Rprotein (“GLP1R humanized mice”). Thirty-one GLP1R humanized mice weredivided into four groups of seven to eight animals. Each group receiveda single subcutaneous injection of isotype control, Q-GLP1-hFc, mAb1, orQ-GLP1-mAb1 at 194 nmol/kg. Mice were bled at fed condition on Days 0,1, 4, 7, 11, 14, 16, 18, and 22 for blood glucose measurements. Mean±SEMof blood glucose levels at each time point was calculated for each groupand shown in Table 2.

TABLE 2 Blood glucose levels Time Isotype Q-GLP1- Q-GLP1- (days) controlhFc mAb1 mAb1 Blood 0 188 ± 6 188 ± 5 188 ± 6 186 ± 8 Glucose 1 185 ± 3184 ± 8 193 ± 8 133 ± 4 (mg/dL) 4 182 ± 9 193 ± 9 180 ± 7 128 ± 5 7 187± 6 199 ± 9 178 ± 7 132 ± 4 11 180 ± 4 192 ± 6 183 ± 8 152 ± 4 14 174 ±6 185 ± 8 184 ± 7 145 ± 4 16 179 ± 6 193 ± 7 183 ± 7 156 ± 4 18 172 ± 7184 ± 8 161 ± 7 154 ± 6 22 174 ± 6 188 ± 7 181 ± 9 171 ± 7

Oral glucose tolerance tests (oGTT) were performed on Day 3 and 9 afterovernight fasting with blood glucose measurements at 0, 15, 30, 60, and120 minutes after a bolus glucose gavarging. Mean±SEM of blood glucoselevels at each time point and glucose area under curve (AUC) werecalculated for each group and shown in Tables 3 and 4.

TABLE 3 Blood glucose levels and glucose AUC on day 3 Time IsotypeQ-GLP1- Q-GLP1- (min) control hFc mAb1 mAb1 Blood 0 143 ± 6 147 ± 5 143± 8 111 ± 5 glucose 15  242 ± 13  252 ± 19  233 ± 16  193 ± 11 (mg/dL)30 233 ± 9 235 ± 8 217 ± 8 160 ± 6 60 187 ± 8 195 ± 7  200 ± 12 129 ± 4120 151 ± 7 160 ± 7  159 ± 10 115 ± 4 Blood glucose 22884 ± 594 23756 ±681 23229 ± 959 16556 ± 386 AUC (mg/dL * 120 min)

TABLE 4 Blood glucose levels and glucose AUC on day 9 Time IsotypeQ-GLP1- Q-GLP1- (min) control hFc m Ab1 mAb1 Blood 0 143 ± 4 151 ± 3 147± 5 116 ± 4 glucose 15  281 ± 17  269 ± 15  253 ± 15  203 ± 13 (mg/dL)30 207 ± 5  228 ± 15  223 ± 14  167 ± 14 60 200 ± 7 187 ± 5 207 ± 8  139± 10 120 155 ± 5  173 ± 11 157 ± 6 131 ± 7 Blood glucose 23589 ± 61023869 ± 795 23959 ± 696 17852 ± 920 AUC (mg/dL * 120 min)

A single administration of Q-GLP1-mAb1 in normoglycemic GLP1R humanizedmice led to significant glucose reductions for 14 days, whereasQ-GLP1-hFc or mAb1 did not affect blood glucose levels (Table 2).Q-GLP1-mAb1 reduced fasting glucose levels and improved glucosetolerance on Day 3 and 9 in the mice, whereas Q-GLP1-hFc and mAb1 didnot. These data suggest that Q-GLP1 or the mAb1 antibody alone does notchange glycemic control, however, a fusion molecule of the two couldexert glucose lowering effects that last for 2 weeks with a singleinjection in normoglycemic animals.

Example 4: Stability of GLP1 Variants

The stability of various GLP1 variants and fusion proteins was tested byincubating them with serum proteases and analyzing the cleaved peptidesby mass spectrometry.

In a first experiment, half μg of each GLP1 fusion protein was addedinto 50 μL naïve mouse serum, respectively. The mixtures were thenincubated at 37° C. for 6 hrs and 24 hrs, respectively. One μL serummixtures at 0 min, 6 hr and 24 hr were loaded onto a Tris-Glycine gel.

In a second experiment, to further differentiate the stabilities, two μgof each GLP1 fusion protein was incubated with 500 ng of recombinanthuman DPP4 (R & D system) in PBS (pH 7.4) at 37° C. for 0 min, 1 hr, 4hrs and 72 hrs, respectively. One fifth of the above mixture (equiv. 400ng of construct) was loaded onto a Tris-Glycine gel.

For each experiment, the gel sections corresponding to each construct'smolecular weight were excised and subject to in-gel trypsin digestion.The excised gel pieces were de-stained in 50:50 acetonitrile: NH₄HCO₃(50 mM), reduced with 65 mM dithiothreitol (Sigma) at 37° C. for 30 minfollowed by alkylation with 135 mM iodoacetamide (Sigma) at roomtemperature in the dark for 30 min. Subsequently, proteins were digestedovernight with sequencing grade modified porcine trypsin (Promega) at37° C. Peptides were extracted twice with extraction buffer (50% ACN, 5%formic acid in H₂O). The extracted peptides from each band were dried tocompletion in SpeedVac and reconstituted with 0.1% tetrafluoroaceticacid (TFA) prior to nanoLC-MS/MS analysis.

The reconstituted peptide mixtures were separated by onlinereverse-phase (RP) nanoscale capillary liquid chromatography(Easy-nLC1000, Thermo Fisher Scientific) and analyzed by electrospraytandem mass spectrometry (Orbitrap Elite, Thermo Fisher Scientific). Thepeptide mixtures were injected onto a 75 μm inner diameter “PepMap RSLC”column (C18, 25 cm, 100 Å, 2 μm, Thermo Fisher Scientific) with a flowrate of 250 nL/min and subsequently eluted with 2% to 35% ACN in 0.1%formic acid in a 60-min gradient. The mass spectrometer was operated indata-dependent mode to automatically switch between MS and MS/MSacquisition. Survey full scan MS spectra (from m/z 350 to 2000) wereacquired in the Orbitrap with a resolution of 120,000. The most intenseions (up to ten), were sequentially isolated for fragmentation in thehybrid ion trap using collision induced dissociation (CID) with anormalized collision energy of 35% at a target value of 5000. Targetions already selected for MS/MS were dynamically excluded for 30 s.

The MS and MS/MS peak lists were extracted and searched against anin-house protein database using ProteomeDiscoverer 1.4 (Thermo FisherScientific). All searches assumed trypsin digestion, and consideredcarboxymethylation of cysteine as a fixed modification and oxidation ofmethionine as a variable modification. A peptide mass tolerance of 10ppm, MS/MS mass tolerance of 0.8 Da, and an allowance for up to 1 misscleavage were used. The extracted ion areas were computed based onextracted ion chromatograms (XIC) using Thermo Xcaliber software (ThermoFisher Scientific).

Results

To characterize each construct's susceptibility to serum enzymecleavages, the intact peptide (N-terminal peptide), cleaved peptide(post-cleavage N-terminal peptide), and one internal reference peptide(a stable peptide unsusceptible to any modification in a construct) foreach construct was monitored by nanoLC-MS/MS. A reduced ratio of theintact peptide vs. the reference peptide and concomitant increased ratioof the cleaved peptide vs. the reference peptide suggestedenzyme-mediated cleavage of a construct over time. Percent cleavage wascalculated using the following formula: 100×area of cleavedpeptide/(area of cleaved peptide+area of uncleaved peptide)

GLP1-hFc and A-GLP1-hFc were cleaved completely by 6 hr, whereasdesH-GLP1-hFc showed noticeable cleavage (2%) by 6 hr. Q-GLP1-hFc andComparator did not show any cleavage after 24 hr incubation (Table 5).

TABLE 5 Percent cleavage of selected GLP1 receptor agonists Cleavage %Cleavage % in 6 hours in 24 hours GLP1-hFc 100% 100% desH-GLP1-hFc   2%  5% Q-GLP1-hFc   0%   0% A-GLP1-hFc 100% 100% Comparator   0%   0%

To further differentiate the stabilities of Q-GLP1-hFc and Comparator,the two constructs were mixed with recombinant human DPP4, and theintact peptide (N-terminal peptide), cleaved peptide (post-cleavageN-terminal peptide), and one internal reference peptide (a stablepeptide unsusceptible to any modification in a construct) for eitherconstruct was monitored by nanoLC-MS/MS.

TABLE 6 Stability of selected GLP1 receptor agonists to DPP4 Cleavage %Cleavage % after 4 hours after 72 hours Q-GLP1-hFc 0%  0% Comparator 4%41%

Comparator showed noticeable cleavage (4%) by 4 hours and over 40%cleavage by 72 hours (Table 6). By contrast, Q-GLP1-hFc did not exhibitany cleavage even after 72-hour incubation with DPP4 at 37° C.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method of lowering blood sugar level in asubject in need thereof, wherein the method comprises administering tothe subject a pharmaceutical composition comprising a therapeuticallyeffective amount of a fusion protein consisting of a glucagon-likepeptide 1 (GLP1) variant and a stabilizing domain, and wherein: (i) theGLP1 variant consists of SEQ ID NO: 6; (ii) the stabilizing domain is anantibody or antigen-binding fragment thereof that binds specifically toGLP1 receptor; and (iii) the fusion protein has enhanced resistance toproteolytic cleavage and/or enhanced blood glucose lowering ability. 2.The method of claim 1, wherein the subject has a disease or disorderselected from the group consisting of diabetes mellitus, obesity,insulin resistance, hypertension, dyslipidemia, Type 2 diabetes, Type 1diabetes, pre-diabetes, cardiovascular disease, atherosclerosis,congestive heart failure, coronary heart disease, arteriosclerosis,peripheral artery disease, stroke, respiratory dysfunction, renaldisease, fatty liver disease, non-alcoholic steatohepatitis (NASH), andmetabolic syndrome.
 3. The method of claim 1, wherein the pharmaceuticalcomposition is administered in combination with a second therapeuticagent or therapy.
 4. The method of claim 3, wherein the secondtherapeutic agent or therapy is selected from the group consisting of aninsulin or insulin analogue, metformin, a thiazolidinedione, asulfonylurea, a biguanide, chlorpropamide, a glinide, an alphaglucosidase inhibitor, nateglinide, a dipeptidyl peptidase 4 (DPP4)inhibitor, pramlintide, sitagliptin, bromocriptine, a sodium glucosecotransporter-2 (SGLT-2) inhibitor, canagliflozin, an anti-hypertensivedrug, a statin, aspirin, dietary modification, exercise, and a dietarysupplement.
 5. The method of claim 1, wherein the pharmaceuticalcomposition is administered subcutaneously, intravenously,intradermally, intraperitoneally, orally, or intramuscularly.